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A couple of weeks ago I had the opportunity to meet with Carlos Ortiz de Solorzano, PhD, staff scientist in the Life Sciences Division, Lawrence Berkeley Laboratory. His research involves doing three-dimensional quantitative analysis of cellular and structural changes in breast tissue, as it progresses from normal to malignant conditions. His lab site reads:
The main goal of my lab is to understand complex biological systems by combining morphological and molecular three-dimensional analysis. For that purpose we use three-dimensional microscopy and quantitative image analysis. Our emphasis is in understanding normal mammary gland development and what goes wrong in breast cancer.
Here’s the way I understand it: Breast tissue is heterogeneous; there are many kinds of cells in a sample
of breast tissue. Furthermore, even cells of the same type may have genetic and phenotypic variances. This is important because a piece of breast tissue is something like a neighborhood; neighboring cells are the environment to each other and they influence each other. The differences among cells are significant in all three dimensions and so is the general architecture of the tissue. A slice through tissue for microscopic inspection that is basically two-dimensional may miss important differences in cells above or below the slice, the third-dimension. It is important to identify cell characteristics cell-by-cell and not assume that cells in the same general area are not significantly different. This is especially true when the tissue starts to be transformed by processes that move cells toward malignancy.
So what Dr. Ortiz de Solorzano has developed is microscopy techniques for looking at blocks of breast tissue that are more than one or two cells thick. He can generate images of tissue in stages of transformation and measure certain changes going left, right, up and down to find distinctions that give a picture (literally, a computer generated image) of a whole structure. He believes that important information about the progression of the disease can be gained by this more whole representation of cell information.
Specifically, Dr. Ortiz de Solorzano has been looking at genetic instability in breast cells as they progress toward malignancy. Basically, genetic instability means that cells undergoing transformation may have abnormal numbers of some chromosomes. Normally, chromosomes come in pairs. If a cell has less than two or more than two of the same chromosome, that generally is a sign of abnormal internal processes, i.e., instability. A working hypothesis is that, as tissues progress toward malignant tumors, the number of cells with abnormal numbers of chromosomes increases. These cells with abnormal chromosome configurations provide a pool of cells that may support the growth or cloning of abnormal cells into malignant masses.
The challenge is that characterizing even small sections of tissue involves finding a way to identify the nuclei of cells and to reliably quantify how many chromosomes they have. Then the cells have to be visualized relative to each other in three dimensions. There are enormous numbers of cells in even a small piece of tissue, and even if you can look at a succession of slices, it’s impossible to put them together in your mind in a precise and reliable way.
Enter clever technique and technology. Dr. Ortiz de Solorzano makes the nuclei of interest identifiable by inducing the nuclei of sample tissues to take up “tags” that attach to genes on two specific chromosomes. The tags have a molecular component that fluoresces when scanned with a certain frequency of light. If a nucleus is normal it will have two glowing dots indicating the normal pair of chromosomes. If it is abnormal (unstable) it will have one or three or more dots. The fluorescent dots in nuclei of cells can be detected by a computer-driven microscope that moves up plane-by-plane in the tissue sample. Then the computer maps the points in three dimensions and computer graphics render an image that looks like the tissue structure—a mammary duct, for example.
With the data from the glowing tags, graphs of the frequency of chromosomes in nuclei can be generated showing in three dimensions the degree of instability in the tissue cells. This creates a sense of the instability and neoplasia for a breast structure or the breast as a whole. These representations add to the ability to characterize how breast cancer progresses.
One interesting additional aspect of the technique and the data collected is to be able to examine the assumption that is often made that malignant tumors are masses of cloned cells with nearly identical genetic characteristics. Dr. Ortiz de Solorzano sees evidence that malignant tumors are heterogeneous with respect to their genetic instability, leading to the likelihood that there are several processes other than cloning going on that affect the genetic characteristics of tumors.
